Challenges in Use of Practice-based Research Networks for a Medical Device Trial to Detect SARS-CoV-2.

INTRODUCTION/OBJECTIVES: Primary care practice-based research networks (PBRNs) participated in a point of care (POC) device study funded by by the National Institutes of Health and led by the University of Massachusetts Chan Medical School (UMass) to speed the development, validation, and commercialization of POC tests to detect SARS-CoV-2. The purposes of this study were to describe the characteristics of participating PBRNs and their respective collaborators in this device trial and describe complications challenging its execution. METHODS: Semi-structured interviews were conducted with lead personnel from participating PBRNs and UMass. RESULTS: Four PBRNs and UMass were invited to participate and 3 PBRNs and UMass participated. This device trial recruited 321 subjects in 6 months; 65 subjects from PBRNs. Each PBRN and the academic medical center site enrolled and recruited subjects differently. Main challenges identified were having adequate clinic personnel to enroll and aid in consent and questionnaire completion, frequently changing inclusion/exclusion criteria, use of the digital electronic data collection platform, and having access to a -80°C freezer to store supplies. DISCUSSION: This trial involved numerous researchers, primary care clinic leaders and staff, and academic center sponsored program staff and attorneys resulting in a resource-intensive endeavor to enroll 65 subjects in the real-world clinical setting of primary care PBRNs with the academic medical center enrolling the rest. Multiple obstacles to standing up the study were encountered by the PBRNS. CONCLUSIONS: Primary care PBRNs rely largely on the goodwill established between academic health centers and participating practices. For future investigations involving device studies, collaborating PBRN leaders should assess whether recruitment criteria may change, obtain detailed lists of equipment needed, and/or know if the study is likely to be halted suddenly to appropriately prepare their member practices.

A live single-cycle RSV vaccine expressing prefusion F protein.

Live-attenuated Respiratory syncytial virus (RSV) vaccines given intranasally have potential to provide comprehensive protection, including lung-resident immunity. It has however proven challenging to impart both sufficient safety and efficacy in a vaccine. To achieve the latter, we used a trans-complementing approach to generate live single-cycle RSV vaccines expressing the prefusion form (preF) of the viral fusion protein (F), either membrane-anchored or secreted. Both viruses were tested for their ability to induce a protective immune response in mice after intranasal prime-boost vaccination. The secreted preF vaccine failed to induce a protective response. The anchored preF vaccine induced anti-preF antibodies and antiviral T cells, and protected mice from lung pathology and viral shedding after challenge. Neither vaccine induced anti-G antibodies, for reasons unknown. In spite of the latter and single-cycle replication, the membrane-anchored preF vaccine was protective and demonstrates potential for development of an efficacious live vaccine with a stable safety phenotype.

Clock-Drawing Test as a Screening Tool for Cognitive Impairment Associated With Fecal Immunochemical Test Collection Errors.

PURPOSE: The purposes of this study were to determine if (1) certain demographic characteristics (potential predictors) of participants, and (2) clock-drawing test results (as a screening test for cognitive impairment) were associated with fecal immunochemical test (FIT) sample collection errors. METHODS: Patients scheduled for an upcoming colonoscopy were asked to collect stool samples using 5 different FITs. Patients completed a questionnaire that included the clock-drawing test. Errors included mistakes or omissions in recording the stool collection date and errors in stool collection. Each clock drawing was scored by 2 reviewers using 2 established methods. RESULTS: Of the 1,448 participants with a clock drawing, 63% were female with a mean age of 63 years. In this population there were 83% White, 6% Black, and 24% Hispanic persons. Cognitive impairment was found in 292 patients by the Mendes-Santos method. Kappa coefficient for the 2 clock-drawing scores was 0.79 (P <.001). The multivariable generalized linear mixed model for FIT collection errors indicated being female (adjusted odds ratio [AOR], 1.64; 95% CI, 1.09-2.48), having an 8th grade or less education (AOR, 3.40; 95% CI, 1.87-6.18), and having an abnormal Mendes-Santos method clock score (AOR, 1.65; 95% CI, 1.08-2.54) were associated with significantly more errors. CONCLUSION: Among the participants who do not have dementia, FIT collection errors were made not only by those who had abnormal clock drawing, but also, by those with normal clock drawings. Subjects being female, having 8th grade education or less, and having an abnormal clock drawing scored by Mendes-Santos's method were associated with FIT collection errors.

Microparticle Delivery of a STING Agonist Enables Indirect Activation of NK Cells by Antigen-Presenting Cells.

Natural killer (NK) cells are an important member of the innate immune system and can participate in direct tumor cell killing in response to immunotherapies. One class of immunotherapy is stimulator of interferon gene (STING) agonists, which result in a robust type I interferon (IFN-I) response. Most mechanistic studies involving STING have focused on macrophages and T cells. Nevertheless, NK cells are also activated by IFN-I, but the effect of STING activation on NK cells remains to be adequately investigated. We show that both direct treatment with soluble STING agonist cyclic di-guanosine monophosphate-adenosine monophosphate (cGAMP) and indirect treatment with cGAMP encapsulated in microparticles (MPs) result in NK cell activation in vitro, although the former requires 100× more cGAMP than the latter. Additionally, direct activation with cGAMP leads to NK cell death. Indirect activation with cGAMP MPs does not result in NK cell death but rather cell activation and cell killing in vitro. In vivo, treatment with soluble cGAMP and cGAMP MPs both cause short-term activation, whereas only cGAMP MP treatment produces long-term changes in NK cell activation markers. Thus, this work indicates that treatment with an encapsulated STING agonist activates NK cells more efficiently than that with soluble cGAMP. In both the in vitro and in vivo systems, the MP delivery system results in more robust effects at a greatly reduced dosage. These results have potential applications in aiding the improvement of cancer immunotherapies.

Trained Clinician's Documentation of Serious Illness Conversations and Use of Billing CPT 99497.

Background: Advance care planning (ACP) involves patients and family members in discussions with clinicians about their values, goals, and preferences regarding future medical care. Objectives: To (1) assess whether an ACP conversation using the Serious Illness Conversation (SIC) was initiated and documented; (2) assess which components of SIC were documented; (3) determine how frequently clinicians trained to use the SIC guide used ACP billing codes during the study time period, (4) determine whether there was a significant difference in mortality risk score according to documentation of each component of the SIC. Methods; Thirteen clinicians at three family medicine offices were trained in the Serious Illness Care Program and asked to document SICs in the electronic medical record (EMR). A retrospective chart review of SIC components was conducted in the EMRs of patients who presumably had ACP conversations initiated by the trained clinicians. Patients were identified using the billing codes for ACP conversations and through referrals from another study that requires clinicians to have ACP conversations with their patients. Pearson chi-square test for categorical variables and t-tests for continuous variables were conducted. Results: A total of 157 patients were included in this study; 131 patients referred from another ACP study and an additional 26 patients using the billing codes of ACP conversations. Through retrospective chart review, the mean age of patients was 72 years and 54 were male. Sixty-two (40%) charts had one or more SIC components documented. "Explore key topics" was documented most frequently for 58 (38%) patients by the 13 participating clinicians. Mean mortality risk score was 10.7 and higher scores were significantly correlated with more SIC components documented (rp = 0.217, P = 0.007). Conclusion: Little use of the SIC guide among trained physicians was found in the EMR. It was expected that provision of an EMR template for documenting the SIC would have facilitated documentation of SICs.

Improving Iowa Research Network Patient Recruitment for an Advance Care Planning Study.

INTRODUCTION/OBJECTIVES: In February 2019, recruitment began in Iowa Research Network offices for a Patient-Centered Outcomes Research Institute (PCORI) funded Advance Care Planning (ACP) study to be conducted in 7 primary care practice-based research networks across the United States and Canada. The main study trained clinicians and nursing staff in serious illness care conversations and requested they refer eligible patients. Eligible patients were those with serious illness or frailty expected to live 1 to 2 years. Clinicians indicated it was difficult to identify eligible patients. This study aimed to find better methods for increasing patient recruitment for the ACP study. METHODS: Research staff brainstormed and implemented strategies to increase patient referrals from clinicians. Participating offices used Epic for their medical record and the Gagne Index was used to generate a list of eligible patients in Epic SlicerDicer. When patients from the Epic SlicerDicer report appeared on the schedule, clinicians and nursing staff were notified that they might be eligible for ACP. Clinicians and nursing staff were asked to complete a survey identifying their perception of implemented strategies. A Wilcoxon signed-rank test was conducted to compare referral numbers before and after the Gagne Index/Epic SlicerDicer intervention. RESULTS: Seven clinicians referred patients prior to and 11 after the Gagne Index/Epic SlicerDicer intervention. Clinicians referred a total of 120 patients; 31 patients prior to and 89 patients after the Gagne Index/Epic SlicerDicer implementation (P = .002). Survey results indicated that several strategies facilitated clinician referrals, including patients identified as potentially appropriate on the schedule, quarterly meetings with researchers, and e-mails with a list of potentially eligible patients. CONCLUSIONS: Notifying clinical staff about potential study participants increased patient referrals in this ACP study. Research staff must have time, funding, and patience to support clinical staff who are expected to refer patients to studies.

Tissue-Resident Memory T Cells in the Lungs Protect against Acute Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young children. The T cell response plays a critical role in facilitating clearance of an acute RSV infection, and memory T cell responses are vital for protection against secondary RSV exposures. Tissue-resident memory (TRM) T cells have been identified as a subset of memory T cells that reside in nonlymphoid tissues and are critical for providing long-term immunity. There is currently limited information regarding the establishment and longevity of TRM T cell responses elicited following an acute RSV infection as well as their role in protection against repeated RSV infections. In this study, we examined the magnitude, phenotype, and protective capacity of TRM CD4 and CD8 T cells in the lungs of BALB/c mice following an acute RSV infection. TRM CD4 and CD8 T cells were established within the lungs and waned by 149 d following RSV infection. To determine the protective capacity of TRMs, FTY720 administration was used to prevent trafficking of peripheral memory T cells into the lungs prior to challenge of RSV-immune mice, with a recombinant influenza virus expressing either an RSV-derived CD4 or CD8 T cell epitope. We observed enhanced viral clearance in RSV-immune mice, suggesting that TRM CD8 T cells can contribute to protection against a secondary RSV infection. Given the protective capacity of TRMs, future RSV vaccine candidates should focus on the generation of these cell populations within the lung to induce effective immunity against RSV infection.

Frontline Science: CD40 signaling restricts RNA virus replication in Mϕs, leading to rapid innate immune control of acute virus infection.

Many acute viral infections target tissue Mϕs, yet the mechanisms of Mϕ-mediated control of viruses are poorly understood. Here, we report that CD40 expressed by peritoneal Mϕs restricts early infection of a broad range of RNA viruses. Loss of CD40 expression enhanced virus replication as early as 12-24 h of infection and, conversely, stimulation of CD40 signaling with an agonistic Ab blocked infection. With peritoneal cell populations infected with the filovirus, wild-type (WT) Ebola virus (EBOV), or a BSL2 model virus, recombinant vesicular stomatitis virus encoding Ebola virus glycoprotein (rVSV/EBOV GP), we examined the mechanism conferring protection. Here, we demonstrate that restricted virus replication in Mϕs required CD154/CD40 interactions that stimulated IL-12 production through TRAF6-dependent signaling. In turn, IL-12 production resulted in IFN-γ production, which induced proinflammatory polarization of Mϕs, protecting the cells from infection. These CD40-dependent events protected mice against virus challenge. CD40-/- mice were exquisitely sensitive to intraperitoneal challenge with a dose of rVSV/EBOV GP that was sublethal to CD40+/+ mice, exhibiting viremia within 12 h of infection and rapidly succumbing to infection. This study identifies a previously unappreciated role for Mϕ-intrinsic CD40 signaling in controlling acute virus infection.

Prognostic Indices for Advance Care Planning in Primary Care: A Scoping Review.

BACKGROUND: Patient identification is an important step for advance care planning (ACP) discussions. OBJECTIVES: We conducted a scoping review to identify prognostic indices potentially useful for initiating ACP. METHODS: We included studies that developed and/or validated a multivariable prognostic index for all-cause mortality between 6 months and 5 years in community-dwelling adults. PubMed was searched in October 2018 for articles meeting our search criteria. If a systematic review was identified from the search, we checked for additional eligible articles in its references. We abstracted data on population studied, discrimination, calibration, where to find the index, and variables included. Each index was further assessed for clinical usability. RESULTS: We identified 18 articles with a total of 17 unique prognostic indices after screening 9154 titles. The majority of indices (88%) had c-statistics greater than or equal to 0.70. Only 1 index was externally validated. Ten indices, 8 developed in the United States and 2 in the United Kingdom, were considered clinically usable. CONCLUSION: Of the 17 unique prognostic indices, 10 may be useful for implementation in the primary care setting to identify patients who may benefit from ACP discussions. An index classified as "clinically usable" may not be easy to use because of a large number of variables that are not routinely collected and the need to program the index into the electronic medical record.

Cytokines and CD8 T cell immunity during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and hospitalization in infants. In spite of the enormous clinical burden caused by RSV infections, there remains no efficacious RSV vaccine. CD8 T cells mediate viral clearance as well as provide protection against a secondary RSV infection. However, RSV-specific CD8 T cells may also induce immunopathology leading to exacerbated morbidity and mortality. Many of the crucial functions performed by CD8 T cells are mediated by the cytokines they produce. IFN-γ and TNF are produced by CD8 T cells following RSV infection and contribute to both the acceleration of viral clearance and the induction of immunopathology. To prevent immunopathology, regulatory mechanisms are in place within the immune system to inhibit CD8 T cell effector functions after the infection has been cleared. The actions of a variety of cytokines, including IL-10 and IL-4, play a critical role in the regulation of CD8 T cell effector activity. Herein, we review the current literature on CD8 T cell responses and the functions of the cytokines they produce following RSV infection. Additionally, we discuss the regulation of CD8 T cell activation and effector functions through the actions of various cytokines.

Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection.

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.

Identification of Novel Respiratory Syncytial Virus CD4+ and CD8+ T Cell Epitopes in C57BL/6 Mice.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection and hospitalization in infants. It is well established that both CD4+ and CD8+ T cells are critical for mediating viral clearance but also contribute to the induction of immunopathology following RSV infection. C57BL/6 mice are often used to study T cell responses following RSV infection given the wide variety of genetically modified animals available. To date, few RSV-derived CD4+ and CD8+ T cell epitopes have been identified in C57BL/6 mice. Using an overlapping peptide library spanning the entire RSV proteome, intracellular cytokine staining for IFN-γ was performed to identify novel CD4+ and CD8+ T cell epitopes in C57BL/6 mice. We identified two novel CD4+ T cell epitopes and three novel CD8+ T cell epitopes located within multiple RSV proteins. Additionally, we characterized the newly described T cell epitopes by determining their TCR Vß expression profiles and MHC restriction. Overall, the novel RSV-derived CD4+ and CD8+ T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses.

Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children, but no vaccine is currently available. Live-attenuated vaccines represent an attractive immunization approach; however, balancing attenuation while retaining sufficient immunogenicity and efficacy has prevented the successful development of such a vaccine. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. The M protein is required for virion assembly following infection of a host cell but is not necessary for either genome replication or gene expression. Therefore, infection with RSV M-null produces all viral proteins except M but does not generate infectious virus progeny, resulting in a single-cycle infection. We evaluated RSV M-null as a potential vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared with its recombinant wild-type control virus (RSV recWT). RSV M-null-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared with mice infected with RSV recWT. Despite its attenuation, RSV M-null infection induced robust immune responses of similar magnitude to that elicited by RSV recWT. Additionally, RSV M-null infection generated serum Ab and memory T cell responses that were similar to those induced by RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, our results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

The CD8 T Cell Response to Respiratory Virus Infections.

Humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. While some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. Despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. Nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. However, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. CD8 T cells are critical for mediating clearance following many acute viral infections in the lung. In addition, memory CD8 T cells are capable of providing protection against secondary infections. Therefore, the combined induction of virus-specific CD8 T cells and antibodies may provide optimal protective immunity. Herein, we review the current literature on CD8 T cell responses induced by respiratory virus infections. Additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on RSV vaccination.

Memory CD8 T cells mediate severe immunopathology following respiratory syncytial virus infection.

Memory CD8 T cells can provide protection from re-infection by respiratory viruses such as influenza and SARS. However, the relative contribution of memory CD8 T cells in providing protection against respiratory syncytial virus (RSV) infection is currently unclear. To address this knowledge gap, we utilized a prime-boost immunization approach to induce robust memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. Unexpectedly, RSV infection of mice with pre-existing CD8 T cell memory led to exacerbated weight loss, pulmonary disease, and lethal immunopathology. The exacerbated disease in immunized mice was not epitope-dependent and occurred despite a significant reduction in RSV viral titers. In addition, the lethal immunopathology was unique to the context of an RSV infection as mice were protected from a normally lethal challenge with a recombinant influenza virus expressing an RSV epitope. Memory CD8 T cells rapidly produced IFN-γ following RSV infection resulting in elevated protein levels in the lung and periphery. Neutralization of IFN-γ in the respiratory tract reduced morbidity and prevented mortality. These results demonstrate that in contrast to other respiratory viruses, RSV-specific memory CD8 T cells can induce lethal immunopathology despite mediating enhanced viral clearance.

Host genetics play a critical role in controlling CD8 T cell function and lethal immunopathology during chronic viral infection.

Effective CD8 T cell responses are vital for the control of chronic viral infections. Many factors of the host immune response contribute to the maintenance of effector CD8 T cell responses versus CD8 T cell exhaustion during chronic infection. Specific MHC alleles and the degree of MHC heterogeneity are associated with enhanced CD8 T cell function and viral control during human chronic infection. However, it is currently unclear to what extent host genetics influences the establishment of chronic viral infection. In order to examine the impact of MHC heterogeneity versus non-MHC host genetics on the development of chronic viral infection, an F1 cross of B10.D2 x B6 (D2B6F1) and BALB.B x BALB/c (BCF1) mice were infected with the clone-13 (Cl-13) strain of lymphocytic choriomeningitis virus (LCMV). Following chronic Cl-13 infection both H-2bxd D2B6F1 and BCF1 mice demonstrated increased viral control compared to homozygous mice. Strikingly, H-2bxd D2B6F1 mice on a C57BL genetic background exhibited mortality following Cl-13 infection. CD8 T cell depletion prevented mortality in Cl-13-infected D2B6F1 mice indicating that mortality was CD8 T-cell-dependent. D2B6F1 mice maintained more CD8 T cell effector cytokine production and exhibited reduced expression of the T cell exhaustion marker PD-1. In addition, D2B6F1 mice also induced a larger Th1 response than BCF1 mice and Cl-13-induced mortality in D2B6F1 mice was also dependent on CD4 T-cell-mediated IFN-γ production. Thus, following a chronic viral infection, increased functionality of the CD8 T cell response allowed for more rapid viral clearance at the cost of enhanced immunopathology dependent on both MHC diversity and the genetic background of the host.

Modulation of the host immune response by respiratory syncytial virus proteins.

Respiratory syncytial virus (RSV) causes severe respiratory disease in both the very young and the elderly. Nearly all individuals become infected in early childhood, and reinfections with the virus are common throughout life. Despite its clinical impact, there remains no licensed RSV vaccine. RSV infection in the respiratory tract induces an inflammatory response by the host to facilitate efficient clearance of the virus. However, the host immune response also contributes to the respiratory disease observed following an RSV infection. RSV has evolved several mechanisms to evade the host immune response and promote virus replication through interactions between RSV proteins and immune components. In contrast, some RSV proteins also play critical roles in activating, rather than suppressing, host immunity. In this review, we discuss the interactions between individual RSV proteins and host factors that modulate the immune response and the implications of these interactions for the course of an RSV infection.